ABSTRACT

Vibrio cholerae, the cause of cholera, has two circular chromosomes. The parAB genes on each V. cholerae chromosome act to control chromosome segregation in a replicon-specific fashion. The chromosome I (ChrI) parAB genes (parAB1) govern the localization of the origin region of ChrI, while the chromosome II (ChrII) parAB genes (parAB2) control the segregation of ChrII. In addition to ParA and ParB proteins, Par systems require ParB binding sites (parS). Here we identified the parS sites on both V. cholerae chromosomes. We found three clustered origin-proximal ParB1 binding parS1 sites on ChrI. Deletion of these three parS1 sites abrogated yellow fluorescent protein (YFP)-ParB1 focus formation in vivo and resulted in mislocalization of the ChrI origin region. However, as observed in a parA1 mutant, mislocalization of the ChrI origin region in the parS1 mutant did not compromise V. cholerae growth, suggesting that additional (non-Par-related) mechanisms may mediate the partitioning of ChrI. We also identified 10 ParB2 binding parS2 sites, which differed in sequence from parS1. Fluorescent derivatives of ParB1 and ParB2 formed foci only with the cognate parS sequence. parABS2 appears to form a functional partitioning system, as we found that parABS2 was sufficient to stabilize an ordinarily unstable plasmid in Escherichia coli. Most parS2 sites were located within 70 kb of the ChrII origin of replication, but one parS2 site was found in the terminus region of ChrI. In contrast, in other sequenced vibrio species, the distribution of parS1 and parS2 sites was entirely chromosome specific.

Bacterial cells must accurately segregate replicated chromosomes to daughter cells to ensure genome integrity over many generations. The mechanisms that mediate chromosome segregation are incompletely understood (41), and there is very little knowledge concerning the mechanisms that are involved in chromosome segregation in bacteria with more than one chromosome. All of the members of the Vibrionaceae and the members of the related family Photobacteriaceae are thought to have two circular chromosomes (36, 42, 43, 50). In Vibrio cholerae, the causative agent of the severe diarrheal disease cholera, chromosome I (ChrI) (2.96 Mb) is larger than chromosome II (ChrII) (1.07 Mb) and contains the majority of the genes considered essential for cell growth; however, the presence of some essential genes on ChrII warrants referring to this replicon as a chromosome (12, 22).

Distinct mechanisms appear to mediate the localization and segregation of the two V. cholerae chromosomes. The origin regions of ChrI and ChrII, oriCIvc and oriCIIvc, respectively, have distinct segregation dynamics. In newborn cells, oriCIvc localizes near the old cell pole, whereas oriCIIvc is found at midcell (15, 16). ChrI undergoes an asymmetric pattern of segregation in which one copy of the newly duplicated oriCIvc remains near the pole, whereas the other copy of oriCIvc traverses across the entire cell to the opposite pole (15-17). In contrast, ChrII undergoes a symmetric pattern of segregation in which the two copies of the duplicated oriCIIvc segregate from the midcell to the cell quarter positions (15, 16). Even though initiation of replication of the two V. cholerae chromosomes is coordinated (13), replicated oriCIvc segregates earlier than replicated oriCIIvc (15, 16). Not much is known about the localization of other chromosomal loci, but it has recently been shown that in newborn cells the terminus region of ChrI (terIvc) is found near the new cell pole, on the side of the cell opposite where oriCIvc is found; terIvc then moves to the midcell region before replication and remains there until just prior to cell division (39; unpublished data). The terminus region of ChrII was observed at midcell, but the timing of its segregation varied (39).

Both of the V. cholerae chromosomes have parAB genes near their replication origins (14, 22). parAB loci are known to be required for partitioning of low-copy-number plasmids (for reviews, see references 11 and 21). Orthologues of parAB genes are also found in many chromosomes, but their roles in chromosome segregation are less clear. Interestingly, the ParA and ParB proteins encoded by the ChrI par locus (parAB1) are similar to other chromosomal ParA and ParB proteins, whereas the ParA and ParB proteins encoded by the ChrII par locus (parAB2) group with plasmid and phage ParA and ParB proteins (22, 51). We recently investigated the roles of the parAB loci in segregation of the two V. cholerae chromosomes. ParAB1 appears to be part of a mitosis-like apparatus that mediates the polar localization and asymmetric segregation pattern of the origin region of ChrI but does not influence segregation of ChrII (17). Although there is mislocalization of oriCIvc in a parA1 mutant, ChrI still successfully segregates to daughter cells in this background, indicating that there are genes other than parAB1 that contribute to ChrI segregation (17, 38). In contrast, parAB2 is required for both localization and segregation of ChrII. In a parAB2 mutant, oriCIIvc is randomly distributed in the cell, and there is a high frequency of ChrII loss (49). The parAB2 mutant does not have a detectable defect in oriCIvc dynamics. Thus, the two V. cholerae parAB loci appear to function in a chromosome-specific fashion.

In plasmids, the partitioning activity of parAB requires a centromere-like, cis-acting site(s) often called parS. ParB binds to parS and is thought to form a nucleoprotein complex that is a target for the ParA ATPase protein. In a few instances, ParA proteins have been shown to form dynamic ATP-dependent polymers, and interactions between ParA polymers and the ParB/parS complex have been proposed to mediate plasmid segregation (3, 4, 11, 21, 29). Unlike parA and parB protein-encoding genes, parS sequences borne on plasmids and phages lack similarity, but they commonly consist of an inverted repeat(s) and/or a direct repeat(s) (21). In contrast, the chromosomal parS sequences that have been identified to date contain an inverted repeat sequence similar to the parS site that was originally identified in the Bacillus subtilis chromosome by Lin and Grossman (2, 10, 19, 23, 24, 26, 30).

Only two studies have elucidated parS sites in bacteria with multiple chromosomes. parS sites were identified on the four replicons of Burkholderia cenocepacia, an opportunistic pathogen with three chromosomes and a low-copy-number plasmid (10). Dubarry et al. used an Escherichia coli plasmid stabilization assay to obtain evidence that the parABS systems on these four replicons do not interact (10). Recently, Saint-Dic et al. identified three putative parS1 sequences on V. cholerae ChrI (38). These three sites are found near oriCIvc and are similar to the B. subtilis parS site (38). Saint-Dic et al. showed that parAB1 could stabilize an ordinarily unstable mini F plasmid harboring any one of three sequences (38). We independently identified these parS1 sites and report here that ParB1 binding to these sites can be detected using a yellow fluorescent protein (YFP)-ParB1 fusion protein or by DNase I protection assays. Furthermore, we show that deletion of these sites from ChrI results in mislocalization of the origin region of ChrI but not a growth defect, phenotypes that were also observed in a parA1 mutant (17, 38). We also identified ParB2 binding sites (parS2). The nucleotide sequence of parS2 differed from that of parS1. Most of the 10 parS2 sites were found to be fairly close to oriCIIvc. Similar patterns of distribution of parS1 and parS2 sites were found in the genomes of V. parahaemolyticus, V. fischeri, V. vulnificus, and Photobacterium profundum, suggesting that in these species the ParAB1 and ParAB2 systems also function in a chromosome-specific manner.

MATERIALS AND METHODS

Bacterial strains and plasmids.Tables 1 and 2 list the key strains and plasmids used in this study. All V. cholerae strains used here were derived from the sequenced El Tor clinical isolate N16961 (22). E. coli strains DH5α, DH5α λpir, and TOP10 were used for DNA cloning. SM10 λpir was used to mobilize DNA into V. cholerae. BL21(DE3) was used for protein purification, and MC1061 was used for the fluorescent focus analyses and the plasmid stability assays.

Deletion and insertion of genes into the N16961 genome were accomplished using pCVD442-based plasmids as described previously (9, 16). In Fig. 3C, some of the plasmids were derived from pLAU43 (25), and the rest of the plasmids were derived from pCR II (Invitrogen, Carlsbad, CA). For most of the plasmids in Fig. 3C, the indicated chromosomal region was initially amplified by PCR. pYB100, which contains the DNA that flanks parS2-A, was constructed by ligating two PCR products that include sequences 5′ and 3′ of parS2-A. Plasmids containing candidate parS sites and used in Tables 3 and 4 were constructed by first synthesizing the appropriate oligonucleotides; then, after annealing, the fragments were inserted into the EcoRI site of pLAU43. When appropriate, the DNA sequences of constructs were verified. Detailed information on the construction of all the plasmids and strains employed here, including the oligonucleotides used, is available upon request.

In vitro DNA binding assays.Binding of ParB1 and ParB2 to parS1 and parS2 sequences, respectively, was assessed by DNase I protection assays using purified His6-tagged proteins. For protein purification, parB1 was cloned into the pET28b expression vector (EMD Biosciences, Madison, WI) under the control of the T7 promoter such that ParB1 had a His6 tag on the N terminus. A 1-liter culture of E. coli BL21(DE3) containing the His6-ParB1 expression vector was grown at 37°C in Luria broth supplemented with 50 μg/ml kanamycin to an optical density at 600 nm of ∼0.6. Expression of His6-ParB1 was induced by addition of 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) for 2 h at 30°C. Cells were harvested and resuspended in 10 ml of buffer A (50 mM NaH2PO4 [pH 8], 0.5 M NaCl, 3 mM dithiothreitol [DTT], 1 mM EDTA, 10% glycerol, 20 mM imidazole) plus 1× Halt protease inhibitor cocktail (Pierce, Rockford, IL). The cells were lysed in a French pressure cell, and insoluble cell debris was sedimented by centrifugation at 10,000 × g for 30 min. The resulting cell lysate was passed over a 1-ml HisTrap FF Crude column (GE Healthcare, Piscataway, NJ) equilibrated with buffer A on a fast protein liquid chromatograph. His6-ParB1 was eluted from the column with a 20-ml linear imidazole gradient (20 to 500 mM imidazole). Fractions containing pure His6-ParB1 (as determined by Coomassie blue staining of a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel) were pooled and dialyzed overnight at 4°C in storage buffer (20 mM Tris-HCl [pH 8], 0.5 M NaCl, 3 mM DTT, 0.1 mM EDTA, 50% glycerol). ParB2-His6 was purified in the same fashion except that the His6 tag was placed on the C terminus.

DNase I protection assays were performed as previously described (6). Briefly, uniquely end-labeled DNA probes were created by PCR with 5′-32P-radiolabeled primer YPR141 (5′-GTGTAGGCTGGAGCTGCTTC-3′) and primer YPR163 (5′-AGTAGCTGACATTCATCCGG-3′) from a plasmid template carrying either the parS1-1 sequence (pYB098) or the parS2-A sequence (pYB086). Binding of His6-ParB1 or ParB2-His6 to radiolabeled DNA probes was performed in the presence of 20 mM Tris-HCl (pH 8), 100 mM NaCl, and 1 mM DTT in a 20-μl mixture for 10 min at room temperature. After binding, 0.1 U of DNase I was added for 30 s at room temperature. The reaction was quenched, and then the mixture was subjected to ethanol precipitation and electrophoresis on an 8% sequencing gel.

Plasmid stability assay.The plasmid stability assay was carried out essentially as described previously (51). E. coli MC1061 cells harboring the test plasmid were grown in LB medium with 20 μg/ml of ampicillin at 37°C, and cells in the mid-log phase were then transferred to fresh LB medium without ampicillin. At various time intervals, aliquots of cells were spread on LB plates and transferred into fresh LB broth to maintain logarithmic growth. To determine the percentage of these cells that contained mini F plasmids, 200 colonies were patched on LB agar plates containing 20 μg/ml of ampicillin at 37°C.

Microscopy.For fluorescence microscopy experiments, cells were cultivated in M9 medium supplemented with glucose (0.2%), Casamino Acids (0.1%), and thiamine (0.1%) at 37°C to the mid-exponential phase. Expression of YFP- and cyan fluorescent protein (CFP)-tagged proteins was induced with 0.08% l-arabinose for 1 h in V. cholerae and with 0.2% l-arabinose for 4 h in E. coli cells. Cells were then transferred to agarose pads on microscope slides. IPTG (0.4 mM) was added to the media to reduce binding of LacI-CFP to lacO arrays as necessary. Images were acquired and processed as previously described (16) or using MetaMorph (Molecular Devices Corporation, Sunnyvale, CA). Matlab software (MathWorks, Natick, MA) was used to detect, count, and measure the localization of LacI-CFP foci as described previously (17). Briefly, sets of phase-contrast and fluorescence images of multiple fields of cells were acquired and processed to detect cell bodies, determine a curved midline for each cell, detect the fluorescent foci, and measure the location of the foci in each cell.

RESULTS

Identification of parS1 sites.In our recent studies, we found that expression of a fluorescent ParB1 derivative (YFP-ParB1) in V. cholerae results in the formation of discrete foci and that these foci are often found very close to the subcellular position of oriCIvc (17). In contrast, when YFP-ParB1 was expressed in E. coli, only diffuse fluorescence and no discrete foci were observed (compare Fig. 1Aa and 1Ba). These observations suggest that there are one or more parS1 sites near oriCIvc and that the E. coli genome lacks similar sites. The lack of observable foci in E. coli also suggests that there are no parS1 sites in the parB1 coding region itself, unlike the case for the B. subtilis spoOJ (parB) sequence (30).

Detection of parS1 sites using fluorescently tagged ParB1. (A) YFP-ParB1 was expressed from pMF392 in wild-type V. cholerae (a) and ΔparS1 V. cholerae (b). (B) YFP-ParB1 was expressed from pMF392 in E. coli containing no other plasmid (a), control vector pWSK129 (b), or pMF301 (c). YFP-ParB1 and LacI-CFP were coexpressed from pYB152 in E. coli harboring pLAU43 (d) and pYB111 (e). YFP-ParB1 is green, and LacI-CFP is magenta. Colocalized fluorescence is white. Bar = 2 μm (C) Schematic representation of the 8-kb chromosomal region that contains the three parS1sites. Arrows represent open reading frames. parS1-1, parS1-2, and parS1-3 sites are indicated by blue bars, and a putative integration host factor binding site is indicated by a black bar. The region deleted in the ΔparS1 mutant (YBB379) is indicated by the gray line. (D) Positions of LacI-CFP foci representing subcellular localization of oriCIvc in wild-type cells (top) (n = 415) and ΔparS1 cells (bottom) (n = 434). Cells containing two foci were analyzed. The focus closest to a pole in each cell is represented by blue dots, and the more distant focus is represented by orange dots. The dashed line shows the mean position of the closest-to-pole focus.

Even though there is considerable diversity among plasmid and phage parS sequences (21), there appears to be some conservation in chromosomal parS sequences (2, 10, 19, 23, 24, 26, 30). In fact, when Lin and Grossman identified the B. subtilis parS sequence in 1998, they found very similar sequences in the then incomplete V. cholerae genome sequence (30). We searched the complete V. cholerae genome for sequences similar to the B. subtilis consensus parS sequence (TGTTNCACGTGAAACA, described in reference 30) and found three sites that have only one mismatch with this consensus sequence. All three sites are clustered within an 8-kb region on ChrI approximately 60 kb from oriCIvc and 80 kb from parAB1 (Fig. 1C and Table 3). Two of the three putative parS sites are found within coding sequences (for a putative thiamine-phosphate pyrophosphorylase [VC0062] and for a putative multidrug resistance protein [VC0069]) (Fig. 1C and Table 3). Interestingly, the three putative parS sites are approximately equally spaced, and there is a putative binding site for integration host factor close to the origin-proximal parS1 site (Fig. 1C).

We utilized fluorescence microscopy to investigate whether these three putative parS sites could bind ParB1 or ParB2 tagged with a fluorescent protein and be detected as discrete fluorescent foci. As mentioned above, when YFP-ParB1 was produced in E. coli, which lacks parS sites, only diffuse fluorescence was observed (Fig. 1Ba). In contrast, when YFP-ParB1 was produced in an E. coli strain harboring pMF301, a derivative of the low-copy-number vector pWSK129 containing the ∼8-kb region with the three parS candidates found on ChrI, several discrete foci were detected (Fig. 1Bc). To strengthen the argument that the YFP-ParB1 foci in Fig. 1Bc correspond to YFP-ParB1 binding to the putative parS sequences in pMF301, we separately introduced each putative 16-bp parS sequence into pLAU43, a plasmid which harbors 240 copies of lacO (25), the binding site for LacI. As expected, when YFP-ParB1 and LacI-CFP were coexpressed in E. coli harboring empty pLAU43, LacI-CFP foci were apparent but YFP-ParB1 fluorescence was diffuse (Fig. 1Bd). In contrast, when any of the three putative ChrI parS sites was introduced into pLAU43, colocalized YFP-ParB1 and LacI-CFP fluorescence was apparent as white foci (Fig. 1Be and Table 3). These observations provide strong support for the idea that each of the three predicted 16-bp parS sites is sufficient to bind ParB1 and that this binding requires no other V. cholerae-specific factors. Finally, DNase I protection assays revealed that purified His6-tagged ParB1 binds to a discrete DNA site that includes the entire predicted 16-bp parS sequence (Fig. 2A), confirming that the predicted parS site is functional for ParB1 binding in the absence of additional factors in vitro. Given these findings, the three ChrI origin-proximal parS sites were designated parS1-1, parS1-2, and parS1-3 (Fig. 1C and Table 3).

DNase I protection assay of parS1 and parS2 DNA. (A) ParB1 bound to parS1-1. The DNase I protection assay was performed with either 0, 8, 16, 32, 63, or 125 nM His6-ParB1 bound to a 5′-32P-labeled 200-bp DNA fragment containing the predicted 16-bp parS1-1 site. (B) ParB2 bound to parS2-A. The DNase I protection assay was performed with either 0, 7, 14, 28, or 56 nM ParB2-His6 bound to a 5′-32P-labeled 200-bp DNA fragment containing the predicted 15-bp parS2-A site. Lane G+A contained the chemical sequencing ladder. The sequence of the protected region is shown on the left in each panel. The vertical lines indicate the DNA regions comprised of either parS1-1 or parS2-A. The arrowheads indicate DNase I-hypersensitive bases detected in the presence of either ParB1 or ParB2.

There appear to be only three parS1 sites in the V. cholerae genome. When two mismatches with the B. subtilis consensus parS sequence were allowed, four additional candidate parS1 sites were found in the V. cholerae genome, three on ChrI and one on ChrII. However, after each of these sites was introduced into pLAU43 and assayed as described above, YFP-ParB1 foci were not detected (Table 3), suggesting that these sequences are not functional ParB1 binding sites. Moreover, deletion of the entire 8-kb region from ChrI that contains the three parS1 sites (Fig. 1C) abrogated YFP-ParB1 focus formation in V. cholerae; when YFP-ParB1 was expressed in the parS1 deletion mutant, only diffuse fluorescence was observed (Fig. 1Ab). Together, these findings lend strong support to the idea that the three ChrI origin-proximal parS1 sites detected by homology with the B. subtilis parS consensus sequence are the only parS1 sites in the V. cholerae genome.

We used a fluorescence reporter-operator system to compare the subcellular localizations of the oriCIvc region in wild-type and ΔparS1 cells. Deletion of the three parS1 sites from ChrI disrupted the polar localization of oriCIvc (Fig. 1D). The subcellular localization of the oriCIvc region in the ΔparS1 cells was very similar to that observed in ΔparA1 cells (17), supporting the idea that parA1 and parS1 function as parts of the same mechanism to promote the tethering of the oriCIvc region to the cell poles. Also like the parA1 mutant, the ΔparS1 mutant did not have a growth defect in rich media, providing additional evidence that the parABS1 system is not the sole mechanism for partitioning ChrI. Growth of the parS1 deletion mutant in M9 media required addition of thiamine, as the deletion in this strain removed several genes important for thiamine biosynthesis. Finally, the ChrI-encoded Par system appears to act in a chromosome-specific fashion, since we found that the three parS1 sites enable focus formation only with YFP-ParB1 and not with YFP-ParB2 (see below).

Identification of ParB2 binding site.As described above for YFP-ParB1, when a YFP-ParB2 fusion protein was expressed in V. cholerae, discrete fluorescent foci were observed (Fig. 3Aa). In contrast, only diffuse fluorescence was observed when YFP-ParB2 was produced in E. coli (Fig. 3Ab). These findings strongly suggest that there are one or more ParB2-specific binding sites (parS2) in the V. cholerae genome and no parS2 sites in the E. coli chromosome or in the parB2 coding region itself. Identifying the parS2 sequence was not as straightforward as determining the parS1 sequence. The difference in the subcellular localizations of YFP-ParB2 and YFP-ParB1 foci (compare Fig. 1Aa with Fig. 3Aa) and the fact that YFP-ParB2 did not form foci (Fig. 3Bb) in E. coli harboring a parS1-bearing plasmid strongly suggested that the parS2 sequence is significantly different from the parS1 sequence. Furthermore, the observation that the subcellular location of the YFP-ParB2 foci in V. cholerae, near the cell's quarter positions, is similar to that of oriCIIvc (15, 16, 38) suggests that at least one or more parS2 sites are located near oriCIIvc.

Detection of parS2 sites using fluorescently tagged ParB2. (A) YFP-ParB2 was expressed from pMF400 in wild-type V. cholerae (a) or in E. coli containing no other plasmid (b), control vector pCRII (c), or pYB040 (d). YFP-ParB2 and LacI-CFP were coexpressed from pYB064 in E. coli containing pLAU43 (e), pYB072 (f), pYB073 (g), pYB090 (h), or pYB100 (i). (B) (a) YFP-ParB1 and LacI-CFP were coexpressed in E. coli harboring pYB090, a plasmid containing lacO and parS2. (b) YFP-ParB2 and LacI-CFP were coexpressed in E. coli harboring pYB111, a plasmid containing lacO and parS1. YFP-ParB1 or YFP-ParB2 is green, and LacI-CFP is magenta. Colocalized fluorescence is white. Representative fields are shown. (C) (Top) Schematic drawing of the region downstream of parB2. Arrows represent open reading frames. The parS2-A site is indicated by a red bar. (Bottom) Chromosomal segments indicated by bars were cloned into pLAU43 (underlined plasmids) or pCR II (plasmids not underlined). pYB090 contains a 15-bp inverted repeat (indicated by facing arrows), and pYB100 contains the regions that flank this repeat. The cloned regions that yielded YFP-ParB2 foci in E. coli are indicated by solid bars; the cloned regions that did not yield YFP-ParB2 foci are indicated by open bars. The common region in the different plasmids that yielded YFP-ParB2 foci is indicated by a gray vertical bar. Detailed information regarding these plasmids is available upon request. (D) Percentage of cells harboring mini F plasmids containing parAB2 genes and parS2-A (pYB145) (▪), parS2-1 (pYB166) (⧫), or parS2-X (pYB220) (⋄). The stability of the control vector, pXX705, without par genes is indicated by the open circles and dotted line. (E) Representative field showing YBB300 cells expressing YFP-ParB2 and LacI-CFP. YFP-ParB2 foci are green, and LacI-CFP foci are magenta. Bar = 2 μm.

Since the V. cholerae ParAB2 proteins are more closely related to plasmid-encoded Par proteins than to other chromosome-encoded ParAB proteins (22, 51) and in several plasmid systems, such as P1 and F, ParB binding sites are located downstream of the parB coding region (for a review, see reference 21), we tested whether parS2 was located downstream of the parB2 gene. We found that YFP-ParB2 foci were observed in E. coli containing pYB040, a pUC-based plasmid with a ∼5.4-kb insert taken from the region downstream of parB2 (Fig. 3Ad). When most of the insert in pYB040 was subcloned into the lacO-bearing vector pLAU43 (resulting in pYB072) and introduced into an E. coli strain producing YFP-ParB2 and LacI-CFP, YFP-ParB2 and LacI-CFP fluorescence colocalized (Fig. 3Af), providing evidence that this fragment contains one or more parS2 sites. As expected, colocalized YFP-ParB2 and LacI-CFP foci were not observed when YFP-ParB2 and LacI-CFP were produced in E. coli containing an empty pLAU43 vector (Fig. 3Ae).

We constructed a series of subclones derived from the 5.4-kb insert in pYB040 to map the region that is sufficient to yield YFP-ParB2 foci in E. coli producing YFP-ParB2. This work revealed that there is a 560-bp region common to all the subclones that yielded YFP-ParB2 foci (Fig. 3C). Analysis of this 560-bp sequence revealed that it contains an inverted repeat sequence, a feature found in several other parS sites (21). This 15-bp sequence contains perfectly matched 7-bp stems separated by one nucleotide (Table 4). A derivative of pLAU43 containing this 15-bp sequence, pYB090, was sufficient to enable YFP-ParB2 to form foci in E. coli (Fig. 3Ah and 3C); in contrast, pYB100, which contains a deletion of the 15-bp sequence from the 560-bp common region, did not enable focus formation (Fig. 3Ai and 3C). These findings strongly suggest that this 15-bp sequence represents the ParB2 binding sequence parS2.

DNase I protection assays were performed with purified His6-tagged ParB2 and parS2 DNA to confirm that ParB2 binds to this sequence. Figure 2B shows that ParB2 binds to a site on the DNA overlapping the parS2 sequence. ParB2 appears to bind parS2 with somewhat greater affinity than ParB1 binds parS1 (Fig. 2). Furthermore, there is evidence that ParB2 spreads along the DNA outside parS2. At higher ParB2 concentrations, we observed a diminution of DNase I hypersensitivity of bases that flank parS2 (Fig. 2B). Additional DNase I protection assays revealed that ParB1 does not bind parS2 and that ParB2 does not bind parS1 (data not shown). We also found that YFP-ParB1 did not make foci in E. coli in the presence of a plasmid carrying parS2 (Fig. 3Ba). Together, these observations indicate that the V. cholerae ParB proteins bind only to their cognate parS sequences.

Identification of multiple parS2 sites across the V. cholerae genome.Two observations suggested that there were multiple parS2 sites in the V. cholerae genome. First, deletion of the parS2 site found downstream from parB2 did not appear to influence V. cholerae growth, even though we recently found that deletion of parB2 and/or parA2 severely reduced V. cholerae growth (49; unpublished data). Second, deletion of this parS2 site did not alter the number or the subcellular localization pattern of YFP-ParB2 foci (data not shown). We searched the V. cholerae genome for sequences similar to the 15-bp parS2 sequence described above (referred to as parS2-A in this paper). We found 14 additional parS2-like sequences when two mismatches with parS2-A were permitted. Nine of these sequences bound ParB2 in the focus formation assay (Table 4). Comparison of the sequences (in Table 4) that enabled YFP-ParB2 focus formation with those that did not suggests that the identities of the first, middle, and last nucleotides in the 15-bp parS2-A sequence are not critical for ParB2 binding. Thus, the consensus sequence for ParB2 binding, at least as revealed by the focus formation assay, is NTTTACANTGTAAAN. In fact, we found that we could alter all three of these positions and the resulting 15 bp sequence still enabled YFP-ParB2 focus formation in E. coli (data not shown).

We searched the V. cholerae genome again for possible additional parS2 sites using the parS2 consensus sequence defined above. Besides the 10 confirmed parS2 sites listed in Table 4, no additional parS2 sites were identified in this search. Nine of the parS2 sites are found on ChrII, and six of these nine sites are within 70 kb of oriCIIvc (Table 4 and Fig. 4A), likely explaining why the subcellular localizations of oriCIIvc and ParB2-YFP appear to be similar. One of the confirmed parS2 sites, parS2-B, is found within rctA, one of the genes required for ChrII replication (14), suggesting the possibility that ParB2 binding to this site could influence ChrII replication. Interestingly, another confirmed parS2 site, parS2-1, is located near the terminus of ChrI (Fig. 4A), raising the possibility that ParB2 could influence the segregation of terIvc.

Bioinformatic assignment of parS1 and parS2 sites in various vibrios. (A) Distribution of parS1 and parS2 sites in the genomes of sequenced vibrios. The locations of parS1 and parS2 sites are indicated by blue and red bars, respectively. The locations of the parAB1 and parAB2 genes are indicated by blue circles and red triangles, respectively. In each case, I refers to ChrI, and II refers to ChrII. (B) Fine distribution of parS2 sites near the oriCII region. The arrows represent annotated open reading frames. Homologous genes are indicated by the same color, and the percent identity to the V. cholerae (Vc) homolog is indicated. The maps are basically drawn to scale; however, some parS1 and parS2 sites are not precisely mapped for clarity. Vp, V. parahaemolyticus; Vv, V. vulnificus (strain YJ016); Vf, V. fischeri; Pp, P. profundum.

Since YFP-ParB2 formed foci in the presence of plasmid-borne parS2-1 in E. coli, we investigated whether YFP-ParB2 foci at the parS2-1 site on the V. cholerae chromosome could be detected. To test this, we examined whether there was colocalization of YFP-ParB2 and LacI-CFP fluorescence in a V. cholerae strain containing an array of lac operators integrated ∼1 kb from the parS2-1 site near terIvc. As previously reported (49), YFP-ParB2 foci were usually seen near the quarter positions of the cell, at the site where oriCIIvc is found (Fig. 3E). LacI-CFP foci, which correspond to the terIvc region, were observed at midcell, and colocalization of YFP-ParB2 and LacI-CFP foci was rarely observed. The lack of detectable YFP-ParB2 foci at midcell is somewhat difficult to explain since YFP-ParB2 formed foci with parS2-1 in E. coli. It is possible that YFP-ParB2 binding to the multiple parS2 sites near the origin of ChrII occurs with greater affinity than binding at parS2-1 (see Discussion).

Functional analysis of the parABS2 system using a heterologous host.We used a mini F plasmid-based stability assay to test whether the parS2 sites, including parS2-1, could serve as functional centromere-like sites with the ParAB2 proteins in E. coli. Several parABS systems have been shown to have partitioning activity using this assay in this heterologous host (2, 10, 19, 38, 49, 51). Introduction of parAB2 along with the parS2-A site into pXX705 (yielding pYB145), an unstable mini F plasmid derivative lacking partitioning genes and sequences, dramatically increased its stability (49) (Fig. 3D), indicating that parABS2 can establish a functional partitioning system in a heterologous host. When parS2-1, the ChrI parS2 site, was used instead of parS2-A, the resulting mini F derivative (pYB166) was also stabilized (Fig. 3D), suggesting that this sequence can function as a centromere-like site nearly as well as parS2-A. We also tested if parS2-X could act as a functional centromere-like site in this assay. This site has two mismatches with the parS2 consensus and did not enable YFP-ParB2 focus formation (Table 4). This site did not stabilize the mini F plasmid (Fig. 3D). Thus, there appears to be a positive correlation between the focus formation assay (Table 4) and the plasmid stabilization assay.

Prediction of parS1 and parS2 sites in other vibrios.Like the V. cholerae genome, the genomes of all of the species in the Vibrionaceae family and the related family Photobacteriaceae are thought to be divided between two chromosomes (36). Furthermore, close homologues of the V. cholerae parAB1 and parAB2 genes are found near the origins of ChrI and ChrII, respectively, in the three other sequenced vibrios (V. parahaemolyticus, V. vulnificus, and V. fischeri) and the closely related bacterium P. profundum (7, 32, 37, 45). We searched for the above-defined consensus parS1 (NGTTNCACGTGAAACN) and parS2 (NTTTACANTGTAAAN) sequences in the complete genome sequences of these four species. All four species had two or more putative origin-proximal parS1 sites and no parS1 sites on their small chromosomes (Fig. 4A). Similarly, all four species had eight or more putative parS2 sites on their ChrIIs; unlike V. cholerae, none of these other organisms had a parS2 site on ChrI. As in V. cholerae, most but not all of the putative parS2 sites identified in these other species were within 70 kb of oriCII. All four organisms contained a parS2 site very close to the ChrII origin of replication, between the parA2 and rctB genes, in the region where rctA is found in V. cholerae (Fig. 4B). Also, a parS2 site was found downstream of parB2 either in the 3′ end of the gene or just downstream from the 3′ end of VCA1112 homologues in these organisms (Fig. 4B). Thus, the putative parS1 and parS2 sites in these four vibrios are entirely chromosome specific, suggesting that in these organisms there is no cross talk between the ParAB1 and ParAB2 systems and their noncognate chromosomes.

DISCUSSION

With one interesting exception, our findings support the notion that the two Par systems in V. cholerae function in a chromosome-specific manner. In the V. cholerae genome, there appear to be only three parS1 sites, all of which are clustered together on ChrI near oriCIvc. Deletion of these three parS1 sites abrogated YFP-ParB1 focus formation in V. cholerae but did not compromise V. cholerae growth. The latter observation likely reflects the existence of additional (non-Par-related) mechanisms that mediate the correct partitioning of the large V. cholerae chromosome. There were no parS1 sites on ChrII, and the parS2 sequence did not enable fluorescently tagged ParB1 to form foci (Fig. 3B); together, these findings suggest that the ParABS1 system acts only on ChrI. The V. cholerae parS2 sequence that we identified was unrelated to parS1. Most of the 10 parS2 sites are located within 70 kb of oriCIIvc, but a few of these sites are more than 100 kb from oriCIIvc and one parS2 site is in the terminus region of ChrI.

The identification of the three parS1 sites near oriCIvc is consistent with our previous findings that the role of ChrI-encoded Par proteins is to ensure the polar localization and segregation of the origin region of ChrI. It is not clear yet if all three V. cholerae parS1 sequences are required for parS1 function in vivo or if one or two sites are sufficient. The lack of growth impairment in the parA1 deletion mutant and the parS1 mutant lacking all three parS1 sites likely reflects the specialized function of the ChrI Par system: localizing the origin region of ChrI to the cell poles (17, 38). Clearly, other mechanisms must exist to partition this chromosome to daughter cells. Given the distribution of the predicted parS1 sites in several other Vibrio species (Fig. 4A), it is likely that all vibrios contain a ChrI Par-mediated mechanism to localize the origin regions of their large chromosomes to the cell poles. It remains to be determined why vibrios contain such an elaborate but nonessential mechanism for controlling the localization and dynamics of the origins of their large chromosomes.

The B. subtilis parS sequence enabled us to rapidly identify the parS sequence on ChrI. The conservation of chromosomal parS sequences in diverse gram-positive and gram-negative bacteria is remarkable and suggests that Par systems appeared early in prokaryote evolution. Although chromosomal parS sequences are conserved, it appears that the function of chromosome-type Par systems varies among diverse bacteria. In B. subtilis, the Par system (Soj and Spo0J) appears to contribute to separation of replicated sister origins during vegetative growth (27). During sporulation, Soj and Spo0J are not essential to localize the origin region to the cell pole (48). In Caulobacter crescentus, where the segregation dynamics of the origin region closely resemble those of oriCIvc, the Par proteins are thought to play a critical role in cytokinesis (33, 40). There is also variation in the number and distribution of conserved chromosomal parS sequences. For example, B. cenocepacia has 2 origin-proximal parS sites on its primary chromosome, whereas Streptomyces coelicolor has 24 parS sites, most of which are found in an origin-proximal region of its 8.67-Mb linear chromosome (10, 23).

Unlike the parS1 sequence, the parS2 sequence was not related to the “universal” chromosomal parS sequence. Phylogenetic analyses of Par protein sequences suggest that ParA2 and ParB2 are more similar to plasmid-encoded Par proteins than to chromosome-encoded Par proteins (22, 51). Unlike the characterized chromosomal parS sites, the sequences and structures of plasmid parS sites are highly variable and often complex (21). Interestingly, there is some similarity of the 15-bp V. cholerae parS2 sequence (NTTTACA-N-TGTAAAN) and the 15-bp OB3 (parS) sequence (TTTTAGC-G-GCTAAAA) in plasmid RK2 (1, 46). Both of these sequences contain a 7-bp inverted repeat separated by a single nucleotide. The parS sites on the secondary chromosomes of Burkholderia species consist of a 16-bp inverted repeat without a spacer (10). The uniqueness of the parS sites on the secondary chromosomes of Vibrio and Burkholderia species compared to the similarity of the parS sites found on the major chromosomes of these species lends credence to the idea that primary and secondary chromosomes have distinct evolutionary histories. However, there is likely selective pressure for divergence of parS sequences on different chromosomes in bacteria with more than one chromosome to avoid partitioning incompatibility between chromosomes.

The locations of the nine parS2 sites that we identified on ChrII generally explain our previous finding that fluorescent ParB2 derivatives can serve as markers of the subcellular location of oriCIIvc (49), since six of the nine sites are located close to oriCIIvc. Currently, it is not known why the three parS2 sites that are fairly distant from oriCIIvc are not detectable as discrete YFP-ParB2 foci. At least three possibilities exist. First, the three origin-distal parS2 sites might be components of a single large ParB2-parS2 nucleoprotein complex formed in vivo. In studies of S. coelicolor, Jakimowicz et al. proposed that many of the 24 parS sites in this organism might be incorporated into a very large nucleoprotein complex (23). A single large ParB2-parS2 complex could facilitate the activity of ParB2 and ParA2 to accurately partition ChrII to daughter cells. However, a single large ParB2-parS2 complex including all of parS2 seems unlikely because it is not consistent with the findings of Srivastava et al. (39), who showed that the localization of the termini of the two V. cholerae chromosomes does not recapitulate that of oriCIIvc. Second, all parS2 sites might be bound by ParB2 in vivo, but not all ParB2-parS2 complexes may be detectable as discrete fluorescent foci. This appears to happen in B. subtilis. Recently, Breier and Grossman (5) used CHIP-chip studies to demonstrate two previously unidentified parS sites found far from oriC. However, only a single fluorescent ParB (Spo0J) focus labeling the origin-proximal region is ordinarily apparent in this organism (18, 31). The reason why these origin-distal sites are not detectable as fluorescent foci is unknown; perhaps, compared to the origin-proximal sites, there are too few fluorescent ParB proteins bound to the origin-distal sites to detect. Finally, ParB2 may not bind all parS2 sites in vivo, perhaps due to ParB2 titration at the origin-proximal parS2 sites or to differing affinities of ParB2 for parS2 sites located in distinct chromosomal contexts. In S. coelicolor, not all 24 parS sites are thought to be bound by ParB in vivo (23).

The parS2 sites located far from oriCIIvc may have functional importance, since we found that all vibrios have parS2 sites located relatively far from their oriCII genes. Systematic deletions of the ChrII parS2 sites will enable study of the necessity for the nine sites in ChrII organization and segregation. Finally, exploration of the biological significance of parS2-1, the parS2 site found near the terminus of ChrI, may provide evidence for a role of the ChrII Par system in segregation of the terminus of ChrI.

The location of a parS2 site adjacent to oriCIIvc in V. cholerae, as well as in all the other sequenced vibrios (Fig. 4B), suggests the possibility that ParB2 binding to this site could influence ChrII replication. In V. cholerae, this parS2-B site lies within rctA, a gene that is thought to encode a nontranslated RNA that regulates oriCIIvc-based replication (14, 44). It seems possible that ParB2 binding to this site could influence transcription of rctA and thereby regulate initiation of ChrII replication. ParB-mediated regulation of replication has been described in B. subtilis and S. coelicolor (23, 27, 28, 35); however, neither of these organisms contains parS sites within the oriC region, so different mechanisms may be involved.

ACKNOWLEDGMENTS

We thank H. Niki for plasmids, B. Karalius for the initial experiments identifying parS1, and Waldor lab members for useful advice and comments on the manuscript. We are grateful to the Tufts-NEMC GRASP Center for medium preparation.

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